The basal forebrain (BF) is a major neuromodulatory nucleus that activates cortex to process relevant sensory stimuli, prepare appropriate motor responses, and engage synaptic plasticity to promote learning. It also provides for increased metabolic demand in cortex by increasing cortical blood flow. Proper cortical function therefore crucially depends on inputs from the BF. Indeed, degeneration of the BF is thought to cause dementia and the cognitive decline associated with Alzheimer's disease. However, the specific cellular targets and physiological effects of the BF to the cortex are not well described. The immediate goal of this proposal is to map the synaptic connectivity and physiological targets of cholinergic BF neurons to better understand the underlying mechanisms of how BF influences cortical activity. Most research on BF has focused on the effects of acetylcholine (ACh), but we have recently shown that cholinergic neurons also have the capacity to cotransmit the inhibitory neurotransmitter GABA. The first part of this proposal (Aim 1) will identify the synaptic connectivity and physiological effect of GABA and ACh cotransmission from cholinergic BF neurons onto both neuronal and non-neuronal targets. Under the mentorship of Dr. Bernardo Sabatini, I will combine my experience with electrophysiology and optogenetics with new training in advanced microscopy and functional imaging of neuronal activity to complete this aim. I will also collaborate with Dr. Chenghua Gu to study how BF signaling regulates cortical blood flow. To facilitate the identification of post-synaptic targets of the BF in a cell-type specific manner, the next portion of the proposed research is dedicated to developing tools for anterograde trans-synaptic tracing (Aim 2). For this aim, I will be trained by my co-mentor, Dr. Stephen Blacklow, in engineering Notch-based synthetic ligand-receptor interactions that report synaptic connections, and by my collaborator, Dr. Constance Cepko, in the use of vesicular stomatitis virus (VSV) for anterograde synaptic tracing. As I transition to independence for Aim 3, I will test the hypothesis that GABA co-release from cholinergic neurons is developmentally regulated and aids in appropriate wiring of cholinergic BF synapses. Finally, I will use intersectional genetics and viral strategies to eliminate either GABA or ACh release from cholinergic BF neurons in order to determine their differential impact on several aspects of cortical activity (Aim 4). This proposal will therefore not only uncover the physiological mechanisms by which BF supports proper cognitive function, but also develop valuable tools that will be generally useful for the study of neural circuitry and explore new ways that neurons may specifically target and regulate multiple neurotransmitters.